Affiliation: Department of Entomology, University of California Davis, Davis, California, United States of America.

ABSTRACT

Background: The mosquito Anopheles funestus is one of the major malaria vector species in sub-Saharan Africa. Olfaction is essential in guiding mosquito behaviors. Odorant-binding proteins (OBPs) are highly expressed in insect olfactory tissues and involved in the first step of odorant reception. An improved understanding of the function of malaria mosquito OBPs may contribute to identifying new attractants/repellents and assist in the development of more efficient and environmentally friendly mosquito controlling strategies.

Methodology: In this study, a large screening of over 50 ecologically significant odorant compounds led to the identification of 12 ligands that elicit significant electroantennographic (EAG) responses from An. funestus female antennae. To compare the absolute efficiency/potency of these chemicals, corrections were made for differences in volatility by determining the exact amount in a stimulus puff. Fourteen AfunOBP genes were cloned and their expression patterns were analyzed. AfunOBP1, 3, 7, 20 and 66 showed olfactory tissue specificity by reverse transcriptase PCR (RT-PCR). Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae. Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds.

Conclusion: This work presents for the first time a study on the odorants and OBPs of the malaria vector mosquito An. funestus, which may provide insight into the An. funestus olfactory research, assist in a comparative study between major malaria mosquitoes An. gambiae and An. funestus olfactory system, and help developing new mosquito control strategies to reduce malaria transmission.

pone-0015403-g008: Binding curves of EAG active compounds to AfunOBP1 and AfunOBP3.Error bars show standard deviation. At pH 7, AfunOBP1 (A) significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3 (B) showed almost no binding activity to any of the selected EAG active compounds.

Mentions:
Here fluorescence binding assay was used to determine the binding activity of AfunOBP1 and AfunOBP3 to the EAG-active compounds. Insect OBPs are involved in the first step of odorant reception where they bind, solubilize and deliver odorant molecules to ORs [10]. We hypothesized that AfunOBP1 and AfunOBP3, two female most abundant olfactory tissue specific OBPs in this study, show binding affinity to some or all of these EAG active odorant compounds. The results showed that AfunOBP1 displays high selectivity towards different EAG-active ligands (Fig. 8A). The ligand with the highest affinity to AfunOBP1 was 2-undecanone, which is also the compound that elicited the highest EAG response per µmol of odorant. 2-Undecanone was first identified from the wild tomato [35] and later used as an insect repellent on mosquitoes such as An. gambiae, Ae. aegypti and ticks [36], [37], [38]. It is still unknown if 2-undecanone is also a repellent to An. funestus. AfunOBP1 also showed binding affinity to nonylacetate, octylacetate as well as 1-octen-3-ol. 1-Octen-3-ol was identified from both cattle [27] and human [25] and has been shown to serve as a powerful attractant for certain species of tsetse flies in the field [27]. Traps with 1-octen-3-ol have resulted in catches of only a few mosquito species, but in combination with CO2, an increase in the collections has been observed [31]. There were no reports on nonyl acetate or octyl acetate to mosquito behavioral study yet. Both are compounds identified from fruits and may be involved in mosquito sugar-feeding behaviors [34]. CquiOBP1, AfunOBP1 orthologous OBP in Cx. quinquefasciatus, was successfully used as molecular target based on its binding affinity to identify several Culex mosquito oviposition attractants in a reverse chemical ecology approach [43]. This study indicated that AfunOBP1 may be used in screening strategies for potential attractants as well as repellents for An. funestus, which may help in mosquito control.

pone-0015403-g008: Binding curves of EAG active compounds to AfunOBP1 and AfunOBP3.Error bars show standard deviation. At pH 7, AfunOBP1 (A) significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3 (B) showed almost no binding activity to any of the selected EAG active compounds.

Mentions:
Here fluorescence binding assay was used to determine the binding activity of AfunOBP1 and AfunOBP3 to the EAG-active compounds. Insect OBPs are involved in the first step of odorant reception where they bind, solubilize and deliver odorant molecules to ORs [10]. We hypothesized that AfunOBP1 and AfunOBP3, two female most abundant olfactory tissue specific OBPs in this study, show binding affinity to some or all of these EAG active odorant compounds. The results showed that AfunOBP1 displays high selectivity towards different EAG-active ligands (Fig. 8A). The ligand with the highest affinity to AfunOBP1 was 2-undecanone, which is also the compound that elicited the highest EAG response per µmol of odorant. 2-Undecanone was first identified from the wild tomato [35] and later used as an insect repellent on mosquitoes such as An. gambiae, Ae. aegypti and ticks [36], [37], [38]. It is still unknown if 2-undecanone is also a repellent to An. funestus. AfunOBP1 also showed binding affinity to nonylacetate, octylacetate as well as 1-octen-3-ol. 1-Octen-3-ol was identified from both cattle [27] and human [25] and has been shown to serve as a powerful attractant for certain species of tsetse flies in the field [27]. Traps with 1-octen-3-ol have resulted in catches of only a few mosquito species, but in combination with CO2, an increase in the collections has been observed [31]. There were no reports on nonyl acetate or octyl acetate to mosquito behavioral study yet. Both are compounds identified from fruits and may be involved in mosquito sugar-feeding behaviors [34]. CquiOBP1, AfunOBP1 orthologous OBP in Cx. quinquefasciatus, was successfully used as molecular target based on its binding affinity to identify several Culex mosquito oviposition attractants in a reverse chemical ecology approach [43]. This study indicated that AfunOBP1 may be used in screening strategies for potential attractants as well as repellents for An. funestus, which may help in mosquito control.

Bottom Line:
In this study, a large screening of over 50 ecologically significant odorant compounds led to the identification of 12 ligands that elicit significant electroantennographic (EAG) responses from An. funestus female antennae.Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae.Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds.

Affiliation:
Department of Entomology, University of California Davis, Davis, California, United States of America.

ABSTRACT

Background: The mosquito Anopheles funestus is one of the major malaria vector species in sub-Saharan Africa. Olfaction is essential in guiding mosquito behaviors. Odorant-binding proteins (OBPs) are highly expressed in insect olfactory tissues and involved in the first step of odorant reception. An improved understanding of the function of malaria mosquito OBPs may contribute to identifying new attractants/repellents and assist in the development of more efficient and environmentally friendly mosquito controlling strategies.

Methodology: In this study, a large screening of over 50 ecologically significant odorant compounds led to the identification of 12 ligands that elicit significant electroantennographic (EAG) responses from An. funestus female antennae. To compare the absolute efficiency/potency of these chemicals, corrections were made for differences in volatility by determining the exact amount in a stimulus puff. Fourteen AfunOBP genes were cloned and their expression patterns were analyzed. AfunOBP1, 3, 7, 20 and 66 showed olfactory tissue specificity by reverse transcriptase PCR (RT-PCR). Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae. Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds.

Conclusion: This work presents for the first time a study on the odorants and OBPs of the malaria vector mosquito An. funestus, which may provide insight into the An. funestus olfactory research, assist in a comparative study between major malaria mosquitoes An. gambiae and An. funestus olfactory system, and help developing new mosquito control strategies to reduce malaria transmission.